Background Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is expressed predominantly in granulocytes. During the 5 years leading up to the present reporting period, we have focused on several areas, including FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. During the immediately antecedent reporting period, we initiated two series of experiments that were completed during the last year. The first was the characterization of mice expressing a truncated form of pyrin. These mice exhibit exaggerated temperature responses to sublethal doses of bacterial endotoxin and increased mortality to higher doses of endotoxin. We identified two mechanisms of endoxin sensitivity, the first involving impairment of caspase-8-dependent apoptosis, and the second involving increased caspase-1 activation and IL-1beta processing, in pyrin-deficient peritoneal macrophages. We hypothesized that pyrin may impair the binding of ASC, an adaptor molecule, to caspase-1, thereby inhibiting IL-1 activation. The second ongoing project has been the analysis of arthritis-associated mutations in PSTPIP1, a protein that we demonstrated to be a pyrin-binding protein. Mutations in PSTPIP1 cause the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), and these mutations lead to increased phosphorylation of PSTPIP1 and consequently increased binding to pyrin. We hypothesized that the increased PSTPIP1-pyrin interaction inhibits pyrin's normal antiinflammatory functions. Results of the Last Year Characterization of pyrin-truncation mice: To complete our initial studies on these animals, we performed a series experiments to test our hypothesis that pyrin and caspase-1 compete for binding to the adaptor protein ASC. We studied glutathione-S-transferase (GST) pull-downs on mixed lysates of NIH-3T3-derived PT67 cells singly transfected with pyrin, caspase-1, or GST-fused murine ASC, and showed that ASC preferentially binds pyrin relative to caspase-1 in this in vitro system. We also performed control experiments demonstrating that the inhibition of apoptosis in pyrin-deficient macrophages is not the direct result of excessive IL-1 beta. A manuscript summarizing the last two years' work on the molecular phenotype of pyrin-truncation mice was published in Molecular Cell in March. Analysis of arthritis-associated mutations in PSTPIP1: To complete these studies, we performed experiments to test the hypothesis that PAPA-associated mutations in PSTPIP1 modulate the effects of pyrin on IL-1beta processing. We utilized a transfection system established by Bertin and colleagues, in which COS-7L cells were transfected with human pyrin, ASC, caspase-1, and mouse IL-1beta expression constructs. With or without cotransfected c-Abl kinase (which phosphorylates PSTPIP1), IL-1beta secretion was accentuated in cells transfected with the A230T and E250Q PSTPIP1 mutants, relative to wild-type. We also studied IL-1 beta production in vitro in monocytes from a PAPA patient with the A230T mutation and active pyoderma gangrenosum compared with healthy controls. Lipopolysaccharide-induced IL-1beta production was substantially increased in the patient relative to controls. Levels of other cytokines, including IL-2, IL-4, IL-5, IL-10, and interferon gamma, were undetectable, whereas IL-6 and IL-12p70, which are inducible by IL-1beta, were also increased. A manuscript analyzing the interaction of pyrin with PSTPIP1, including the effects of PAPA-associated mutations, is in press in the Proceedings of the National Academy of Sciences. Interaction of human pyrin with caspase-1: In our analysis of pyrin-deficient mice, we found that murine pyrin interacts with caspase-1 not only through the ASC adaptor protein, but by directly binding caspase-1. We went on to show that human pyrin coimmunoprecipitates caspase-1, but not caspases 2-10. The p20p10 catalytic domain of caspase-1 was both necessary and sufficient for the interaction with human pyrin, and the C-terminal B30.2 region of pyrin was the major interacting domain of pyrin. Three major FMF-associated B30.2-domain FMF mutations, M680I, M694V, and V726A, were associated with a substantially decreased interaction with caspase-1, relative to the wild-type pyrin. Cotransfection of these 3 pyrin mutants with caspase-1 resulted in the appearance of an approximately 50 kDa pyrin fragment. This fragment was markedly diminished in cells transfected with caspase-1 deletion mutants lacking an intact catalytic domain, or in the presence of the caspase-1-specific inhibitor ZWEHD-FMK. These data led us to hypothesize that pyrin can be directly cleaved by caspase-1, and that the proportion of pyrin undergoing cleavage is increased in the presence of the 3 aforementioned FMF-associated mutations. To test the first hypothesis, we synthesized radiolabeled pyrin in an in vitro transcription and translation system. Radiolabeled pyrin was cleaved by purified human caspase-1 in a dose-dependent manner. In other experiments, cleaved fragments were electrophoresed, blotted, eluted, and subjected to Edman degradation sequencing. The sequence was the same as the pyrin sequence from residues 331 to 340, indicating that caspase-1 cleaves pyrin between aspartate-330 and serine-331. Conclusions and Significance Our data on pyrin-truncation mice indicate an important role for pyrin in the regulation of the innate immune response through its effects on macrophage apoptosis and IL-1 production. We have also defined the interaction between pyrin and PSTPIP1, and have provided evidence linking PAPA syndrome, an inherited autoinflammatory disorder of the skin and joints, with abnormalities in pyrin-PSTPIP1 binding. By placing PSTPIP1 in the pyrin pathway of inflammation, we have demonstrated a direct role for PSTPIP1 phosphorylation in the control of pyrin's actions, and have added a fifth clinical disorder to the list of pyrin-related autoinflammatory diseases. Our data suggest that other PSTPIP1-associated proteins may also indirectly influence the pyrin pathway, and that mutations in these proteins may define yet other inflammatory disorders. Our data on the interaction of pyrin with caspase-1 indicate that, at least under some conditions, pyrin is cleaved by caspase-1, and that FMF-associated mutations in the B30.2 domain of pyrin lead to markedly increased caspase-1-mediated cleavage. These data suggest that increased caspase-1-mediated cleavage of pyrin may be an important mechanism of disease in FMF, and may in some way account for the apparent selective advantage in FMF carriers. During the next year, our objectives will be 1) to continue patient-oriented studies aimed at gene-discovery and elucidating disease mechanisms; 2) to extend studies in animal models, particularly a recently-developed line of pyrin-null mice; 3) to continue functional studies of pyrin, particularly with regard to the biologic relevance of caspase-1-mediated cleavage; 4) to undertake structural studies of pyrin and related proteins; 5) to study gene expression profiles of patients with FMF during and between attacks; and 6) to undertake clinical trials of IL-1 inhibition in FMF.